The long term objectives of this work are to use vaccinia virus as a model to study transcriptional processes and to understand, at the molecular level, the regulation of the switch from early to late transcription in the life cycle of the virus. An in vitro transcription system which will use vaccinia virus late genes as templates has been developed and fractionated by chromatography into components which can be functionally assayed by an in vitro complementation assay. RNA polymerase is one component of this system, however, additional factors, present only in vaccinia virus infected cells, must be present to allow transcription of the late viral genes. The specific goals of this work, therefore, are: (1) to further purify the factors required for late transcription, (2) to map and characterize these factors, and (3) to determine the mechanism of late transcription initiation. Purification will be done by standard chromatographic techniques following the activity of the factors using the in vitro complementation assay. Mapping will be done by obtaining antibodies to screen an expression library, by mapping late defective mutants, or by characterizing the proteins identified by a recently developed in vivo complementation assay. Once mapped, the DNA sequence of the targeted region will be determined and the regulation of the identified gene(s) investigated. Later, mutagenesis studies will be performed to study structure-function relationships of these factors. Understanding the character and regulation of the factors necessary for late transcription will help to realize the long term goal of understanding the mechanism of the early to late switch. Once the factors which are responsible for activating late genes are characterized, it should be possible to alter the virus in such a way as to make it a more efficient vector for expression of foreign genes for vaccine use.